I am trying to measure Calcium influx in TRPV4 stimulated/unstimulated RAW 264.7 cells using Fura-2 AM (incubation for 45mins at 37degrees) and a plate reader. I was hoping that when I get high values for 340nm, the intesity values for 380 have to decrease, but this in not the case. When 340 goes up, also the 380 does. Is this normal? What am I doing wrong?
Thank you!
I've been successful with a million cells with some cell types I've been able to incubate with fura at least 1 hour with half a million. It's better to try different numbers of cells as well as incubation times for example some cancer cell lines needs 2 hours for fura dowloading for measuarments.
All bests!
Even though I have tried with different cell counts and also different time intervals for dye loading but no significant changes were seen.
Timur Gainutdinov
Fura – 2FF (AM)
(M.w. 1024, P = 50µg, Store at -20oC, Prod. F-1044)
Fura-2FF (AM) soluble in DMSO (The AM ester form should be prepared in high-quality anhydrous DMSO)
[100 µM] is stock solution (50mg / 50µl DMSO at -20oC)
[ ]end = 80 µM (D.G.Nicholls, JBC, 2003, 278, 19062-19070)
Kd= 5.5 µM (D.G.Nicholls, JBC, 2003, 278, 19062-19070)
For cells: Kd = for Ca2+ is 135 nM in Mg2+-free buffers and 224 nM in the presence of 1 mM Mg2+ (Molecular Probes)
Experimental procedure
Brain mitochondria (0.1 mg of protein) were incubated in 25µl of buffer (A) containing:
sucrose 250 mM,
MOPS 10 mM,
fura-2FF AM 80µM
BSA 16 µM,
pH 7.2
on ice for 30 min and then at room temperature for 30 min. The mitochondria were then centrifuged for 1 min at 10000 x g, the pellet resuspended in 1ml of buffer and recentrifuged. The pellet was finally resuspended in 20 µl buffer A and used immediately.
Setup
Wavelength setup.
Excitation (nm)
Emission (nm)
Wavelength 1
340
510
Wavelength 2
380
510
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